Reverse genetics using non-endogenous pol i promoters

ABSTRACT

Expression of a transgene is driven in a host cell using a pol I promoter which is not endogenous to an organism from the same taxonomic order from which the host cell is derived.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.13/320,902, claiming an international filing date of May 21, 2010; whichis the National Stage of International Patent Application No.PCT/IB2010/01332, filed May 21, 2010; which claims the benefit of U.S.Provisional Patent Application No. 61/216,919, filed May 21, 2009, eachof which is hereby incorporated by reference in its entirety.

SUBMISSION OF SEQUENCE LISTING AS ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 223002122801SEQLIST.txt,date recorded: Mar. 17, 2015, size: 22 KB).

TECHNICAL FIELD

This invention is in the field of reverse genetics. Furthermore, itrelates to manufacturing vaccines for protecting against variousviruses.

BACKGROUND ART

Reverse genetics permits the recombinant expression and manipulation ofRNA viruses m cell culture. It is a powerful tool in virology andvaccine manufacture because it allows rapid production of recombinantviruses (including reassortants) and/or their mutation. The methodinvolves transfecting host cells with one or more expression constructsthat encode the viral genome and isolating the virus from the cells. Forexample, references 1 and 2 describe a method in which the influenzagenomic RNA is expressed in canine cells using the canine pol Ipromoter. Other sources have reported the expression of influenzagenomic RNA in human cells using the human pol I promoter.

One significant drawback of the methods of the prior art is that pol Ipromoters are highly species specific. For example, it has been reportedthat the human pol I promoter is active only in primate cells [3], andsimilarly that expression in canine cells would require the canine pol Ipromoter. Thus, where a virus needs to be grown in a cell line for whichthe endogenous pol I promoter has not been characterized, it has beennecessary to use two different cell types for rescuing and growing thevirus. However, it is desirable to avoid the use of multiple cell linesas this has the advantage, for example, that competing culture selectionpressures can be avoided. The use of a single cell line for all steps ofvaccine production also facilitates regulatory approval. Thus there is acontinued need in the art to provide alternative methods for practisingreverse genetics.

SUMMARY OF PREFERRED EMBODIMENTS

The inventors have now surprisingly discovered that it is possible todrive expression of a transgene in a host cell using a pol I promoterwhich is not endogenous to an organism from the same taxonomic orderfrom which the host cell is derived.

In one embodiment, the invention provides a host cell comprising one ormore expression construct(s) wherein expression of a RNA molecule fromthe construct(s) is controlled by a pol I promoter which is notendogenous to the host cell's order. These host cells can be used inexpression systems of the invention.

The invention further provides a process for RNA expression in a hostcell, comprising the steps of (i) preparing an expression constructwherein expression of a transgene of interest is driven by a pol Ipromoter from a first organism and (ii) introducing the expressionconstruct of step (i) into a host cell, wherein the host cell is from adifferent taxonomic order from the first organism.

In a further embodiment, the invention provides a method for producing arecombinant virus wherein the virus is produced using a host cell of theinvention.

The invention also provides a method of preparing a virus (e.g. forformulation into a vaccine), comprising steps of (i) producing arecombinant virus using a host cell of the invention (ii) infecting aculture host with the virus obtained in step (i), (iii) culturing theculture host from step (ii) in order to produce virus; and (iv)purifying the virus obtained in step (iii). To provide a method ofpreparing a vaccine, the method can then include the further step of (v)formulating the virus into a vaccine.

In addition to the non-endogenous pol I promoter(s) which are introducedas discussed above, the host cell will include endogenous pol Ipromoters. The non-endogenous pol I promoter(s) drive(s) expression ofnon-endogenous RNA, in particular viral RNA, in the cell. The inventionthus provides a cell having at least one endogenous pol I promoter whichcontrol(s) expression of endogenous rRNA and at least one non-endogenouspol I promoter which control(s) expression of a viral RNA or thecomplement thereof.

The invention also provides a DNA expression construct encoding both aprotein-coding mRNA and a viral RNA, wherein (i) the codon usage in theDNA for the protein-coding mRNA is optimised for canine cells and (ii)the viral RNA is under the control of a primate pol I promoter. Thecanine cells are ideally MDCK cells and the primate promoter is ideallya human pol I promoter. Expression of the protein-coding mRNA may beunder the control of a pol II promoter optimised for canine cells.

Expression Constructs

The present inventors have surprisingly discovered that it is possibleto drive RNA expression in a cell using a pol I promoter from anorganism which is in a different taxonomic order from the cell. Thus thepol I promoter is not endogenous to an organism from the same taxonomicorder from which the cell is derived. The term “order” refers toconventional taxonomic ranking, and examples of orders are primates,rodentia, carnivora, marsupialia, cetacean, etc. Humans and chimpanzeesare in the same taxonomic order (primates), but humans and dogs are indifferent orders (primates vs. carnivora).

Thus in a first aspect, the invention provides a host cell comprisingone or more expression construct(s) wherein expression of a RNA moleculefrom the construct(s) is driven by a pol I promoter which is notendogenous to the host cell's order.

In one embodiment, the host cell is a non-primate cell and the pol Ipromoter is a primate pol I promoter. In a specific embodiment, the hostcell is a non-primate cell and the promoter is a human promoter. In afurther embodiment, the host cell is a non-human cell and the pol Ipromoter is a human pol I promoter. In an alternative embodiment, thepol I promoter is a non-canine pol I promoter and the host cell is acanine cell. In a preferred embodiment, the host cell is a canine celland the promoter is a primate pol I promoter. In a further preferredembodiment, the pol I promoter is a human promoter and the host cell isa canine cell (such as a MDCK cell). This embodiment is preferred as thehuman pol I promoter is well characterised and canine cells are oftenused for the production of vaccines.

The expression constructs used in the host cells may be uni-directionalor bi-directional expression constructs. Where a host cell expressesmore than one transgene (whether on the same or different expressionconstructs) it is possible to use uni-directional and/or bi-directionalexpression.

Bi-directional expression constructs contain at least two promoterswhich drive expression in different directions (i.e. both 5′ to 3′ and3′ to 5′) from the same construct. At least one of the promoters is anon-endogenous pol I promoter as discussed herein. The two promoters canbe operably linked to different strands of the same double stranded DNA.Preferably, one of the promoters is a non-endogenous pol I promoter andat least one of the other promoters is a pol II promoter. This is usefulas the pol I promoter can be used to express uncapped cRNAs while thepol II promoter can be used to transcribe mRNAs which can subsequentlybe translated into proteins, thus allowing simultaneous expression ofRNA and protein from the same construct. The pol II promoter may beendogenous or non-endogenous. Where more than one expression constructis used within an expression system, the promoters may be a mixture ofendogenous and non-endogenous promoters provided that at least one ofthe promoters is a non-endogenous pol I promoter that can driveexpression in the host cell.

The expression construct will typically include an RNA transcriptiontermination sequence. The termination sequence may be an endogenoustermination sequence or a termination sequence which is not endogenousto the host cell. Suitable termination sequences will be evident tothose of skill in the art and include, but are not limited to, RNApolymerase I transcription termination sequence, RNA polymerase IItranscription termination sequence, and ribozymes. Furthermore, theexpression constructs may contain one or more polyadenylation signalsfor mRNAs, particularly at the end of a gene whose expression iscontrolled by a pol II promoter.

An expression system may contain at least two, at least three, at leastfour, at least five, at least six, at least seven, at least eight, atleast nine, at least ten, at least eleven or at least twelve expressionconstructs.

An expression construct may be a vector, such as a plasmid or otherepisomal construct. Such vectors will typically comprise at least onebacterial and/or eukaryotic origin of replication. Furthermore, thevector may comprise a selectable marker which allows for selection inprokaryotic and/or eukaryotic cells. Examples of such selectable markersare genes conferring resistance to antibiotics, such as ampicillin orkanamycin. The vector may further comprise one or more multiple cloningsites to facilitate cloning of a DNA sequence.

As an alternative, an expression construct may be a linear expressionconstruct. Such linear expression constructs will typically not containany amplification and/or selection sequences. However, linear constructscomprising such amplification and/or selection sequences are also withinthe scope of the present invention. An example of a method using suchlinear expression constructs for the expression of influenza virus isdescribed in reference 4.

Expression constructs of the invention can be generated using methodsknown in the art. Such methods were described, for example, in reference5. Where the expression construct is a linear expression construct, itis possible to linearise it before introduction into the host cellutilising a single restriction enzyme site. Alternatively, it ispossible to excise the expression construct from a vector using at leasttwo restriction enzyme sites. Furthermore, it is also possible to obtaina linear expression construct by amplifying it using a nucleic acidamplification technique (e.g. by PCR).

The expression constructs of the invention can be introduced into hostcells using any technique known to those of skill in the art. Forexample, expression constructs of the invention can be introduced intohost cells by employing electroporation. DEAE-dextran, calcium phosphateprecipitation, liposomes, microinjection, or microparticle-bombardment.

Where the expression host is a canine cell, such as a MDCK cell line,protein-coding regions may be optimised for canine expression e.g. usinga promoter from a wild-type canine gene or from a canine virus, and/orhaving codon usage more suitable for canine cells than for human cells.For instance, whereas human genes slightly favour UUC as the codon forPhe (54%), in canine cells the preference is stronger (59%). Similarly,whereas there is no majority preference for Ile codons in human cells,53% of canine codons use AUC for Ile. Canine viruses, such as canineparvovirus (a ssDNA virus) can also provide guidance for codonoptimisation e.g. 95% of Phe codons in canine parvovirus sequences areUUU (vs. 41% in the canine genome), 68% of Ile codons are AUU (vs. 32%),46% of Val codons are GUU (vs. 14%), 72% of Pro codons are CCA (vs.25%), 87% of Tyr codons are UAU (vs. 40%). 87% of His codons are CAU(vs. 39%), 92% of Gin codons are CAA (vs. 25%), 81% of Glu codons areGAA (vs. 40%), 94% of Cys codons are UGU (vs. 42%), only 1% of Sercodons are UCU (vs. 24%), CCC is never used for Phe and UAG is neverused as a stop codon. Thus protein-coding genes can be made more likegenes which nature has already optimised for expression in canine cells,thereby facilitating expression.

Reverse Genetics

The expression constructs and host cells described above areparticularly suitable for producing recombinant virus strains throughreverse genetics techniques. The techniques can be used for theproduction of a wide variety of RNA viruses, including positive-strandRNA viruses [6.7], negative-strand RNA viruses [8,9] and double-strandedRNA viruses [10]. Thus, in a further aspect, the present inventionprovides a method for producing a recombinant virus wherein the virus isproduced using an expression system as described above.

Known reverse genetics systems involve expressing DNA molecules whichencode desired viral RNA (vRNA) molecules from pol I promoters,bacterial RNA polymerase promoters, bacteriophage polymerase promoters,etc. Furthermore, where a virus requires certain proteins to form aninfectious virus, systems also provide these proteins e.g. the systemfurther comprises DNA molecules that encode viral proteins such thatexpression of both types of DNA leads to assembly of a completeinfectious virus.

Where reverse genetics is used for the expression of vRNA, it will beevident to the person skilled in the art that precise spacing of thesequence elements with reference to each other is pivotal for thepolymerase to initiate replication. It is therefore important that theDNA molecule encoding the viral RNA is positioned correctly between thepol I promoter and the termination sequence, but this positioning iswell within the capabilities of those who work with reverse geneticssystems.

Generally, reverse genetics is suitable for expression of any viruseswhich are known to require production of genomic RNA during theirlife-cycle. Such viruses include, but are not limited to,positive-strand and negative-strand RNA viruses, such as those describedbelow. Preferably, the virus is an orthomyxovirus, e.g., an influenzavirus. The methods of the invention are further suitable fornon-segmented as well as segmented viruses.

Where the virus is a positive-strand RNA virus it is often sufficient totransfect a cell with an expression construct comprising the viralgenome. For example, the transfection of plasmids containing thepoliovirus genome resulted in the recovery of infectious poliovirus[6,7]. Reverse genetics for negative-strand RNA viruses has presentedmore challenges as the antisense viral RNA is usually non-infective andrequires an RNA polymerase to complete the life cycle. Thus, the viralpolymerase must be supplied, either as protein or as a gene for in situprotein expression.

Where the virus requires a protein for infectivity, it is generallypreferred to use bi-directional expression constructs as this reducesthe total number of expression constructs required by the host cell.Thus, the method of the invention may utilise at least onebi-directional expression construct wherein a gene or cDNA is locatedbetween an upstream pol II promoter and a downstream non-endogenous polI promoter. Transcription of the gene or cDNA from the pol II promoterproduces capped positive-sense viral mRNA which can be translated into aprotein, while transcription from the non-endogenous pol I promoterproduces negative-sense vRNA. The bi-directional expression constructmay be a bi-directional expression vector.

In order to produce a recombinant virus, a cell must express allsegments of the viral genome which are necessary to assemble a virion.DNA cloned into the expression constructs of the present inventionpreferably provides all of the viral RNA and proteins, but it is alsopossible to use a helper virus to provide some of the RNA and proteins,although systems which do not use a helper virus are preferred. Wherethe virus is a non-segmented virus this can usually be achieved byutilising a single expression construct in the method of the invention,even though it is also within the scope of the invention to express theviral genome of non-segmented viruses using more than one expressionconstruct. Where the virus is a segmented virus, the viral genome isusually expressed using more than one expression construct in the methodof the invention. However, it is also envisioned to combine one or moresegments or even all segments of the viral genome on a single expressionconstruct.

Methods of the invention are particularly suitable for the production ofreassortant virus strains. The technique can use in vitro manipulationof plasmids to generate combinations of viral segments, to facilitatemanipulation of coding or non-coding sequences in the viral segments, tointroduce mutations, etc. The use of the expression system for theproduction of reassortant virus strains is preferred as this cansignificantly decrease the time needed to obtain a reassortant seedvirus which is particularly beneficial in situations where a rapidproduction of vaccine is needed to counteract an epidemic. Thus, it ispreferred that the method of this aspect of the invention uses one ormore expression constructs that express viral genes from or derived fromat least two different wild type strains.

In some embodiments an expression construct will also be included whichleads to expression of an accessory protein in the host cell. Forinstance, it can be advantageous to express a non-viral serine protease(e.g. trypsin) as part of a reverse genetics system.

When the expression constructs of the invention are used for theexpression of influenza A viral segments, it is possible to generate theexpression construct by introducing the influenza A viral segment intoan expression construct comprising a negative selection marker (forexample, ccdB) and the highly conserved influenza A virus gene termini[11]. The advantage of this is that no restriction sites are requiredand that any influenza A viral segment can be cloned provided it hastermini which are complementary to the gene termini on the expressionconstruct.

Cells

The present invention can be practised with any eukaryotic orprokaryotic cell that can produce the virus of interest. The inventionwill typically use a cell line although, for example, primary cells maybe used as an alternative. The cell will typically be mammalian.Suitable mammalian cells include, but are not limited to, hamster,cattle, primate (including humans and monkeys) and dog cells. Variouscell types may be used, such as kidney cells, fibroblasts, retinalcells, lung cells, etc. Examples of suitable hamster cells are the celllines having the names BHK21 or HKCC. Suitable monkey cells are e.g.African green monkey cells, such as kidney cells as in the Vero cellline [12-14]. Suitable dog cells are e.g. kidney cells, as in the CLDKand MDCK cell lines.

Further suitable cells include, but are not limited to: CHO; 293T; BHK;MRC 5; PER.C6 [15]: FRhL2; WI-38; etc. Suitable cells are widelyavailable e.g. from the American Type Cell Culture (ATCC) collection[16], from the Coriell Cell Repositories [17], or from the EuropeanCollection of Cell Cultures (ECACC). For example, the ATCC suppliesvarious different Vero cells under catalogue numbers CCL 81, CCL 81.2,CRL 1586 and CRL-1587, and it supplies MDCK cells under catalogue numberCCL 34. PER.C6 is available from the ECACC under deposit number96022940.

Preferred cells (particularly for growing influenza viruses) for use inthe invention are MDCK cells [18-20], derived from Madin Darby caninekidney. The original MDCK cells are available from the ATCC as CCL 34.It is preferred that derivatives of these cells or other MDCK cells areused. Such derivatives were described, for instance, in reference 18which discloses MDCK cells that were adapted for growth in suspensionculture (‘MDCK 33016’ or ‘33016-PF’, deposited as DSM ACC 2219; see alsoref. 18). Furthermore, reference 21 discloses MDCK-derived cells thatgrow in suspension in serum free culture (‘B-702’, deposited as FERMBP-7449). In some embodiments, the MDCK cell line used may betumorigenic. It is also envisioned to use non-tumorigenic MDCK cells.For example, reference 22 discloses non tumorigenic MDCK cells,including ‘MDCK-S’ (ATCC PTA-6500), ‘MDCK-SF101’ (ATCC PTA-6501),‘MDCK-SFL02’ (ATCC PTA-6502) and ‘MDCK-SF103’ (ATCC PTA-6503). Reference23 discloses MDCK cells with high susceptibility to infection, including‘MDCK.5F1’ cells (ATCC CRL 12042).

It is possible to use a mixture of more than one cell type to practisethe methods of the present invention. However, it is preferred that themethods of the invention are practised with a single cell type e.g. withmonoclonal cells. Preferably, the cells used in the methods of thepresent invention are from a single cell line. Furthermore, the samecell line may be used for rescuing the virus and for any subsequentpropagation of the virus.

Preferably, the cells are cultured in the absence of serum, to avoid acommon source of contaminants. Various serum-free media for eukaryoticcell culture are known to the person skilled in the art (e.g. Iscove'smedium, ultra CHO medium (BioWhittaker), EX-CELL (JRH Biosciences)).Furthermore, protein-free media may be used (e.g. PF-CHO (JRHBiosciences)). Otherwise, the cells for replication can also be culturedin the customary serum-containing media (e.g. MEM or DMEM medium with0.5% to 10% of fetal calf serum).

The cells may be in adherent culture or in suspension.

Screening for Suitable Cell Lines

Suitable cells for use in accordance with the present invention arewidely available. Furthermore, it is possible to screen for furthercells using techniques commonly known in the art. Screening for suitablecells may be necessary, for example, where a new pol I promoter isidentified and where it is desirable to find cell lines which willsupport expression by the new promoter. Likewise, where a new cell isisolated, it may be necessary to confirm which pol I promoters can driveexpression in it.

Suitable techniques for screening cells will be evident to those ofskill in the art. For example, a reporter gene can be cloned undercontrol of the pol I promoter of interest and the construct can betransfected into the cell line which is to be screened. In suchexperiments, cells transfected with a construct that contains thereporter gene but lacks a promoter sequence can be used as a control.Thus, where the expression of the gene in a test sample (e.g. cellscontaining a transgene under control of the pol I promoter of interest)is significantly higher than the expression in the control (e.g. cellscontaining the same transgene as the test sample but where the transgenedoes not contain a promoter to drive expression of the transgene), thecell line is suitable for use with that promoter according to theinvention. Expression of the transgene can be measured, for example, byreverse transcribing the transgene RNA and subjecting the obtained cDNAto real-time PCR. Alternatively, it is also possible to clone a reportergene (e.g. GFP, YFP, luc etc.) in antisense direction under control ofthe pol I promoter. A transcript from such a construct may then betranscribed into mRNA by a viral polymerase and subsequently betranslated into a protein. Thus, any cell which expresses the reportergene can be easily identified by the presence of the reporter geneproduct.

It is further possible to adapt cells in which a foreign pol I would notnormally drive expression to obtain cells in which the pol I promotercan drive expression. This can be achieved, for example, by subjectingthe cells to growth conditions which would not normally be suitable forthem. For example, a cell line which would normally grow only adherentlycan be held artificially in suspension and the cells which continue togrow under these conditions can be propagated further. Alternatively, itis possible to adherently culture cells that would normally grow insuspension, e.g. by using high binding plastic culture vessels or byadding serum to the culture. Similarly, it is possible to grow cellswhich normally require serum for their growth under serum-freeconditions or, conversely, to expose cells which are adapted toserum-free growth to serum. The selected cells can then be assayed foractivity of the pol I promoter, as described earlier. Further suitablegrowth parameters which can be altered in this manner will be apparentto those of skill in the art and include, but are not limited to,temperature, pH, pO₂, serum concentration, etc. Furthermore, the cellscan be subjected to physical or chemical treatments, such as UVradiation or to chemical mutagens. Likewise, it is possible to screenfor new properties of cells which have merely been passaged under normalculture conditions.

For example, reference 18 describes a method in which MDCK cells(usually adherent) were adapted to growth in suspension under serum-freeconditions. Starting cells were cultivated in serum-free medium inroller bottles under conditions which are normally used to cultivatecells that grow in suspension. Following several passages under theseselective conditions, several cell lines were obtained that could growin suspension in serum-free medium. One example is the 33016 cell line(deposited as DSM ACC 2219). The inventors have demonstrated that ahuman pol I promoter can drive expression of a reporter gene in theseMDCK cells.

RNA Polymerase I Promoters

Most reverse genetics methods use expression vectors which comprise aRNA polymerase I (RNA pol I) promoter to drive transcription of viralgenomic RNA. The pol I promoter gives a transcript with unmodified 5′and 3′ ends which is necessary for full infectivity of many viruses e.g.influenza.

Natural pol I promoters are bipartite, having two separate regions: thecore promoter and the upstream promoter element (UPE). Although thisgeneral organisation is common to pol I promoters from most species,however, the actual sequences of the promoters vary widely. The corepromoter surrounds the transcription startpoint, extending from about−45 to +20, and is sufficient to initiate transcription. The corepromoter is generally GC rich. Although the core promoter alone issufficient to initiate transcription, the promoter's efficiency is verymuch increased by the UPE. The UPE typically extends from about −180 to−107 and is also GC rich. The activity of the promoter may be furtherenhanced by the presence of distal enhancer-like sequences, which mightfunction by stabilizing the pre-initiation complex.

The sequence of the pol I promoter has been identified in a variety ofspecies, including human, dog and chicken. The invention uses a pol Ipromoter which is not endogenous to an organism in the same taxonomicorder as the host cell. The terms “endogenous” and “non-endogenous” arethus used in relation to the host cell and the pol I promoter which ispresent in an expression construct. The inter-species sequence variationin pol I promoters means that it is simple to determine whether anyparticular pol I promoter in a cell is endogenous or non-endogenous.Thus the invention may utilise the human, dog or chicken pol I promoterfor RNA expression in a host cell which is not derived from the sametaxonomic order as the pol I promoter (e.g. a primate pol I promoter ina canine host cell). Sequence comparisons, either in silico orexperimental, can be used to confirm the organism from which anyparticular pol I promoter is derived e.g. FIG. 10 shows an alignment ofthe canine and human pol promoters up to the transcription start site,with <60% sequence identity.

Expression constructs of the invention include at least one corepromoter; preferably they also include at least one UPE, and they mayalso include one or more enhancer elements. It is also possible to usethe fragments of natural promoters, provided that these fragments caninitiate transcription. For example, FIG. 3 shows the sequence of thefull-length canine pol I promoter (SEQ ID NO: 3) and various fragmentswhich are sufficient to drive expression of a transgene (see also

FIG. 4 and SEQ ID NOs: 4 and 5). Furthermore, FIG. 2 shows the sequenceof the human pol I promoter (SEQ ID NO: 1) and a fragment of it whichalone is sufficient for transgene expression in the host cell (see alsoFIG. 5 and SEQ ID NO: 2).

A human pol I promoter which can be used according to the invention maycomprise the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a variantthereof. Where a canine promoter is used according to the invention, itmay comprise the sequence of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5,or a variant thereof.

The pol I promoter may comprise (i) a sequence having at least p %sequence identity to any of SEQ ID NOs: 1 to 5, and/or (ii) a fragmentany of SEQ ID NOs: 1 to 5, provided that the promoter has the ability toinitiate and drive transcription of an operatively linked RNA-encodingsequence in a host cell of interest. The value of p may be 75, 80, 85,90, 95, 96, 97, 98, 99 or more. The fragment may itself be of sufficientlength to drive expression (e.g. SEQ ID NO: 4 is a fragment of SEQ IDNO: 3) or the fragment may be joined to other sequences and thiscombination will drive expression. The ability of such pol I promotersto drive expression in a host cell of interest can readily be assessede.g. using the assays described above with an antisense reporter geneunder control of the promoter.

Virus Preparation

In a further aspect, the present invention provides a method ofpreparing a virus for vaccine manufacture, comprising steps of (i)producing a recombinant virus as described herein (ii) infecting aculture host with the virus obtained in step (i), (iii) culturing thehost from step (iii) in order to produce virus; (iv) purifying the virusobtained in step (iii) and (optionally) (v) formulating the virus into avaccine.

Where cells are used as a culture host in this aspect of the invention,it is known that cell culture conditions (e.g. temperature, celldensity, pH value, etc.) are variable over a wide range subject to thecell line and the virus employed and can be adapted to the requirementsof the application. The following information therefore merelyrepresents guidelines.

As mentioned above, cells are preferably cultured in serum-free orprotein-free media.

Multiplication of the cells can be conducted in accordance with methodsknown to those of skill in the art. For example, the cells can becultivated in a perfusion system using ordinary support methods likecentrifugation or filtration. Moreover, the cells can be multipliedaccording to the invention in a fed-batch system before infection. Inthe context of the present invention, a culture system is referred to asa fed-batch system in which the cells are initially cultured in a batchsystem and depletion of nutrients (or part of the nutrients) in themedium is compensated by controlled feeding of concentrated nutrients.It can be advantageous to adjust the pH value of the medium duringmultiplication of cells before infection to a value between pH 6.6 andpH 7.8 and especially between a value between pH 7.2 and pH 7.3.Culturing of cells preferably occurs at a temperature between 30 and 40°C. In step (iii), the cells are preferably cultured at a temperature ofbetween 30° C. and 36° C. or between 32° C. and 34° C. or at 33° C. Thisis particularly preferred where the method of the invention is used toproduce influenza virus, as it has been shown that incubation ofinfected cells in this temperature range results in production of avirus that results in improved efficacy when formulated into a vaccine[24].

The oxygen partial pressure can be adjusted during culturing beforeinfection preferably at a value between 25% and 95% and especially at avalue between 35% and 60%. The values for the oxygen partial pressurestated in the context of the invention are based on saturation of air.Infection of cells occurs at a cell density of preferably about 8-25 x10⁵ cells/mL in the batch system or preferably about 5-20 x 10⁶ cells/mLin the perfusion system. The cells can be infected with a viral dose(MOI value, “multiplicity of infection”; corresponds to the number ofvirus units per cell at the time of infection) between 10⁻⁸ and 10,preferably between 0.0001 and 0.5.

Virus may be grown on cells in adherent culture or in suspension.Microcarrier cultures can be used. In some embodiments, the cells maythus be adapted for growth in suspension.

The methods according to the invention also include harvesting andisolation of viruses or the proteins generated by them. During isolationof viruses or proteins, the cells are separated from the culture mediumby standard methods like separation, filtration or ultrafiltration. Theviruses or the proteins are then concentrated according to methodssufficiently known to those skilled in the art, like gradientcentrifugation, filtration, precipitation, chromatography, etc., andthen purified. It is also preferred according to the invention that theviruses are inactivated during or after purification. Virus inactivationcan occur, for example, by β-propiolactone or formaldehyde at any pointwithin the purification process.

The viruses isolated in step (i) can also be grown on eggs in step (ii).The current standard method for influenza virus growth for vaccines usesembryonated SPF hen eggs, with virus being purified from the eggcontents (allantoic fluid). It is also possible to passage a virusthrough eggs and subsequently propagate it in cell culture.

Viruses

The methods of the invention may be practised with any virus which canbe expressed by reverse generics in a cell. Such viruses can besegmented or non-segmented viruses. Furthermore, the virus can be apositive-strand RNA virus or a negative-strand virus. In a furtherembodiment, the virus may also be a double-stranded RNA virus.

Where the virus is a negative-strand RNA virus, the virus may be from afamily selected from the group consisting of Paramyxoviridae,Pneumovirinae, Rhabdoviridae, Filoviridae, Bornaviridae,Orthomyxoviridae, Bunyaviridae, or Arenaviridae. Furthermore, the virusmay be a virus from a genus selected from the group consisting ofParamyxovirus, Orthomyxovirus, Respirovirus. Morbillivirus, Rubulavirus,Henipaviras, Avulavirus, Pneumovirus, Metapneumovirus, Vesiculovirus,Lyssavirus. Ephemerovirus, Cytorhabdovirus, Nucleorhabdovirus,Novirhabdovirus, Marburgvirus, Ebolavirus, Bornavirus, Influenzavirus A,Influenzavirus B, Influenzavirus C, Thogotovirus, Isavirus,Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus, Tospovirus,Arenavirus, Ophiovirus, Tenuivirus, or Deltavirus. In specificembodiments, the negative-strand RNA virus is selected from the groupconsisting of Sendai virus, Measles virus, Mumps virus, Hendra virus,Newcastle disease virus, Human respiratory syncytial virus, Avianpneumovirus, Vesicular stomatitis Indiana virus, Rabies virus, Bovineephemeral fever virus, Lettuce necrotic yellows virus, Potato yellowdwarf virus, Infectious hematopoietic necrosis virus, Lake Victoriamarburgvirus, Zaire ebolavirus, Borna disease virus. Influenza virus,Thogoto virus, Infectious salmon anemia virus, Bunyamwera virus, Hantaanvirus, Dugbe virus, Rift Valley fever virus, Tomato spotted wilt virus,Lymphocytic choriomeningitis virus, Citrus psorosis virus, Rice stripevirus, and Hepatitis delta virus. In preferred embodiments, the virus isan influenza virus (see below).

Where the virus is a positive-strand RNA virus, the virus may be from afamily selected from the group consisting of Arteriviridae,Coronaviridae, Picornaviridae and Roniviridae. Furthermore, the virusmay be a virus from a genus selected from the group consisting ofArterivirius, Coronavirus, Enterovirus, Torovirus, Okavirus, Rhinovirus,Hepatovirus, Cardiovirus, Aphthovirus, Parechovirus, Erbovirus,Kobuvirus and Teschovirus. In specific embodiments, the virus isselected from the group consisting of severe acute respiratory syndrome(SARS) virus, polio virus, Human enterovirus A (HEV-A), Humanenterovirus B (HEV-B), Human enterovirus C, Human enterovirus D,Hepatitis A and Human rhinovirus A and B.

Where the virus is a double-stranded RNA virus, the virus may be from afamily selected from the group consisting of Birnaviridae, Cystoviridae,Hypoviridae, Partitiviridae. Reoviridae and Totiviridae. Furthermore,the virus may be a virus from a genus selected from the group consistingof Aquabirnavirus, Avibirnavirus, Entomobirnavirus, Cystovirus,Partitivirus. Alphacryptovirus, Betacryptovirus, Aquareovirus,Coltivirus, Cypovirus, Fijivirus, Idnoreovirus, Mycoreovirus, Orbivirus,Orthoreovirus, Oryzavirus. Phytoreovirus, Rotavirus and Scadornavirus.

The present invention is particularly suitable for viruses which undergorapid mutation and where the recombinant approach allows for a morerapid isolation of the virus which can then be further propagated toobtain suitable vaccines. Therefore, in a preferred embodiment the virusis influenza.

Influenza Viruses

Influenza viruses are particularly suitable for use in the methods ofthe present invention, particularly influenza A viruses and influenza Bviruses, as reverse genetics for this virus has been well characterized.Influenza viruses are segmented negative strand RNA viruses. Influenza Aand B viruses have eight segments, whereas influenza C virus has seven.The virus requires at least four viral proteins (PB1, PB2, PA andnucleoprotein) to initiate replication and transcription.

Reverse genetics for influenza A and B viruses can be practised with 12plasmids to express the four required proteins and all eight genomesegments. To reduce the number of constructs, however, a plurality ofRNA polymerase I transcription cassettes (for viral RNA synthesis) canbe included on a single plasmid (e.g. sequences encoding 1, 2, 3, 4, 5,6, 7 or all 8 influenza vRNA segments), and a plurality ofprotein-coding regions with RNA polymerase II promoters on anotherplasmid (e.g. sequences encoding 1, 2, 3, 4, 5, 6, 7 or 8 influenza mRNAtranscripts) [25]. It is also possible to include one or more influenzavRNA segments under control of a pol I promoter and one or moreinfluenza protein coding regions under control of another promoter, inparticular a pol II promoter, on the same plasmid. As described above,this is preferably done by using bi-directional plasmids. Preferredaspects of the reference 25 method involve: (a) PB1, PB2 and PAmRNA-encoding regions on a single plasmid; and (b) all 8 vRNA encodingsegments on a single plasmid. Including the neuraminidase (NA) andhemagglutinin (HA) segments on one plasmid and the six other segments onanother plasmid is particularly preferred as newly emerging influenzavirus strains usually have mutations in the NA and/or HA segments.Therefore, in this embodiment, only the vector comprising the HA and NAsequence needs to be replaced.

Preferred expression systems for influenza A viruses encode genomesegments derived from a plurality of different wild-type strains. Thesystem may encode 1 or more (e.g. 1, 2, 3, 4, 5 or 6) genome segmentsfrom a PR/8/34 strain (A/Puerto Rico/8/34), but usually this/these willnot include the PR/8/34 HA segment and usually will not include thePR/8/34 NA segment. Thus the system may encode at least one of segmentsNP, M, NS, PA, PB1 and/or PB2 (possibly all six) from PR/8/34.

Other useful expression systems for influenza A viruses may encode 1 ormore (e.g. 1, 2, 3, 4, 5 or 6) genome segments from an AA/6/60 influenzavirus (A/Ann Arbor/6/60), but usually this/these will not include theAA/6/60 HA segment and usually will not include the AA/6/60 NA segment.Thus the system may encode at least one of segments NP, M, NS, PA, PB1and/or PB2 (possibly all six) from AA/6/60.

The system may encode 1 or more genome segments from anA/California/4/09 strain e.g. the HA segment and/or the NA segment.Thus, for instance, the HA gene segment may encode a H1 hemagglutininwhich is more closely related to SEQ ID NO: 6 than to SEQ ID NO: 7 (i.e.has a higher degree sequence identity when compared to SEQ ID NO: 6 thanto SEQ ID NO: 7 using the same algorithm and parameters). SEQ ID NOs: 6and 7 are 80% identical. Similarly, the NA gene may encode a N1neuraminidase which is more closely related to SEQ ID NO: 8 than to SEQID NO: 9. SEQ ID NOs: 8 and 9 are 82% identical.

Expression systems for influenza B viruses may encode genome segmentsderived from a plurality of different wild-type strains. The system mayencode 1 or more (e.g. 1, 2, 3, 4, 5 or 6) genome segments from aAA/1/66 influenza virus (B/Ann Arbor/l/66), but usually this/these willnot include the AA/I/66 HA segment and usually will not include theAA/1/66 NA segment. Thus the system may encode at least one of segmentsNP, M, NS, PA, PB and/or PB2 from AA/1/66.

Viral segments and sequences from the A/PR/8/34, AA/6/60, AA/l/66,A/Chile/1/83 and A/California/04/09 strains are widely available. Theirsequences are available on the public databases e.g. GI:89779337,GI:89779334, GI:89779332, GI:89779320, GI:89779327, GI:89779325,GI:89779322, GI:89779329.

A reverse genetics system for influenza virus may include an expressionconstruct which leads to expression of an accessory protein in the hostcell. For instance, it can be advantageous to express a non-viral serineprotease (e.g. trypsin).

Vaccine

The method of the third aspect of the invention utilises virus producedaccording to the method to produce vaccines.

Vaccines (particularly for influenza virus) are generally based eitheron live virus or on inactivated virus. Inactivated vaccines may be basedon whole virions, ‘split’ virions, or on purified surface antigens.Antigens can also be presented in the form of virosomes. The inventioncan be used for manufacturing any of these types of vaccine.

Where an inactivated virus is used, the vaccine may comprise wholevirion, split virion, or purified surface antigens (for influenza,including hemagglutinin and, usually, also including neuraminidase).Chemical means for inactivating a virus include treatment with aneffective amount of one or more of the following agents: detergents,formaldehyde, β-propiolactone, methylene blue, psoralen,carboxyfullerene (C60), binary ethylamine, acetyl ethyleneimine, orcombinations thereof. Non-chemical methods of viral inactivation areknown in the art, such as for example UV light or gamma irradiation.

Virions can be harvested from virus-containing fluids, e.g. allantoicfluid or cell culture supernatant, by various methods. For example, apurification process may involve zonal centrifugation using a linearsucrose gradient solution that includes detergent to disrupt thevirions. Antigens may then be purified, after optional dilution, bydiafiltration.

Split virions are obtained by treating purified virions with detergents(e.g. ethyl ether, polysorbate 80, deoxycholate, tri-N-butyl phosphate.Triton X-100, Triton N101, ceryltrimethylammonium bromide. Tergitol NP9,etc.) to produce subvirion preparations, including the ‘Tween-ether’splitting process. Methods of splitting influenza viruses, for exampleare well known in the art e.g. see refs. 26-31, etc. Splitting of thevirus is typically carried out by disrupting or fragmenting whole virus,whether infectious or non-infectious with a disrupting concentration ofa splitting agent. The disruption results in a full or partialsolubilisation of the virus proteins, altering the integrity of thevirus. Preferred splitting agents are non-ionic and ionic (e.g.cationic) surfactants e.g. alkylglycosides, alkylthioglycosides, acylsugars, sulphobetaines, betains, polyoxyethylenealkylethers,N,N-dialkyl-Glucamides, Hecameg, alkylphenoxy-polyethoxyethanols, NP9,quaternary ammonium compounds, sarcosyl, CTABs (cetyl trimethyl ammoniumbromides), tri-N-butyl phosphate, Cetavion, myristyltrimcthylammoniumsalts, lipofectin, lipofectamine, and DOT-MA, the octyl- or nonylphenoxypolyoxyethanols (e.g. the Triton surfactants, such as Triton X-100 orTriton N101), polyoxyethylene sorbitan esters (the Tween surfactants),polyoxyethylene ethers, polyoxyethlene esters, etc. One useful splittingprocedure uses the consecutive effects of sodium deoxycholate andformaldehyde, and splitting can take place during initial virionpurification (e.g. in a sucrose density gradient solution). Thus asplitting process can involve clarification of the virion-containingmaterial (to remove non-virion material), concentration of the harvestedvirions (e.g. using an adsorption method, such as CaHPO₄ adsorption),separation of whole virions from non-virion material, splitting ofvirions using a splitting agent in a density gradient centrifugationstep (e.g. using a sucrose gradient that contains a splitting agent suchas sodium deoxycholate), and then filtration (e.g. ultrafiltration) toremove undesired materials. Split virions can usefully be resuspended insodium phosphate-buffered isotonic sodium chloride solution. Examples ofsplit influenza vaccines are the BEGRIVAC™, FLUARIX™, FLUZONE™ andFLUSHIELD™ products.

The method of the invention may also be used to produce live vaccines.Such vaccines are usually prepared by purifying virions fromvirion-containing fluids. For example, the fluids may be clarified bycentrifugation, and stabilized with buffer (e.g. containing sucrose,potassium phosphate, and monosodium glutamate). Various forms ofinfluenza virus vaccine are currently available (e.g. see chapters 17 &18 of reference 32). Live viruse vaccines include MedImmune's FLUMIST™product (trivalent live virus vaccine).

Purified influenza virus surface antigen vaccines comprise the surfaceantigens hemagglutinin and, typically, also neuraminidase. Processes forpreparing these proteins in purified form are well known in the art. TheFLUVIRIN™, AGRIPPAL™ and INFLUVAC™ products are influenza subunitvaccines.

Another form of inactivated antigen is the virosome [33](nucleic acidfree viral-like liposomal particles). Virosomes can be prepared bysolubilization of virus with a detergent followed by removal of thenucleocapsid and reconstitution of the membrane containing the viralglycoproteins. An alternative method for preparing virosomes involvesadding viral membrane glycoproteins to excess amounts of phospholipids,to give liposomes with viral proteins in their membrane.

The virus may be attenuated. The virus may be temperature-sensitive. Thevirus may be cold-adapted. These three features are particularly usefulwhen using live virus as an antigen.

HA is the main immunogen in current inactivated influenza vaccines, andvaccine doses are standardised by reference to HA levels, typicallymeasured by SRID. Existing vaccines typically contain about 15 μg of HAper strain, although lower doses can be used e.g. for children, or inpandemic situations, or when using an adjuvant. Fractional doses such as½ (i.e. 7.5 μg HA per strain), ¼ and ⅛ have been used, as have higherdoses (e.g. 3x or 9x doses [34,35]). Thus vaccines may include between0.1 and 150 μg of HA per influenza strain, preferably between 0.1 and 50μg e.g. 0.1-20 μg, 0.1-15 μg, 0.1-10 μg, 0.1-7.5 μg, 0.5-5 μg, etc.Particular doses include e.g. about 45, about 30, about 15, about 10,about 7.5, about 5, about 3.8, about 3.75, about 1.9, about 1.5, etc.per strain.

For live vaccines, dosing is measured by median tissue cultureinfectious dose (TCID₅₀) rather than HA content, and a TCID₅₀ of between10⁶ and 10⁸ (preferably between 10^(6.5)-10^(7.5)) per strain istypical.

Influenza strains used with the invention may have a natural HA as foundin a wild-type virus, or a modified HA. For instance, it is known tomodify HA to remove determinants (e.g. hyper-basic regions around theHA1/HA2 cleavage site) that cause a virus to be highly pathogenic inavian species. The use of reverse genetics facilitates suchmodifications.

Influenza virus strains for use in vaccines change from season toseason. In inter-pandemic periods, vaccines typically include twoinfluenza A strains (H1N1 and H3N2) and one influenza B strain, andtrivalent vaccines are typical. The invention may also use pandemicviral strains (i.e. strains to which the vaccine recipient and thegeneral human population are immunologically naïve, in particular ofinfluenza A virus), such as H2, H5. H7 or H9 subtype strains, andinfluenza vaccines for pandemic strains may be monovalent or may bebased on a normal trivalent vaccine supplemented by a pandemic strain.Depending on the season and on the nature of the antigen included in thevaccine, however, the invention may protect against one or more of HAsubtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14,H15 or H16. The invention may protect against one or more of influenza Avirus NA subtypes N1, N2, N3, N4, N5, N6, N7, N8 or N9.

As well as being suitable for immunizing against inter-pandemic strains,the compositions of the invention are particularly useful for immunizingagainst pandemic or potentially-pandemic strains. The characteristics ofan influenza strain that give it the potential to cause a pandemicoutbreak are: (a) it contains a new hemagglutinin compared to thehemagglutinins in currently-circulating human strains, i.e. one that hasnot been evident in the human population for over a decade (e.g. H2), orhas not previously been seen at all in the human population (e.g. H5, H6or H9, that have generally been found only in bird populations), suchthat the human population will be immunologically naïve to the strain'shemagglutinin; (b) it is capable of being transmitted horizontally inthe human population; and (c) it is pathogenic to humans. A virus withH5 hemagglutinin type is preferred for immunizing against pandemicinfluenza, such as a H5N1 strain. Other possible strains include H5N3,H9N2, H2N2, H7N1 and H7N7, and any other emerging potentially pandemicstrains. The invention is particularly suitable for protecting againstpotential pandemic virus strains that can or have spread from anon-human animal population to humans, for example a swine-origin H1N1influenza strain. The invention is then suitable for vaccinating humansas well as non-human animals.

Other strains whose antigens can usefully be included in thecompositions are strains which are resistant to antiviral therapy (e.g.resistant to oseltamivir [36] and/or zanamivir), including resistantpandemic strains [37].

Compositions of the invention may include antigen(s) from one or more(e.g. 1, 2, 3, 4 or more) influenza virus strains, including influenza Avirus and/or influenza B virus. Where a vaccine includes more than onestrain of influenza, the different strains are typically grownseparately and are mixed after the viruses have been harvested andantigens have been prepared. Thus a process of the invention may includethe step of mixing antigens from more than one influenza strain. Atrivalent vaccine is typical, including antigens from two influenza Avirus strains and one influenza B virus strain. A tetravalent vaccine isalso useful [38], including antigens from two influenza A virus strainsand two influenza B virus strains, or three influenza A virus strainsand one influenza B virus strain.

Pharmaceutical Compositions

Vaccine compositions manufactured according to the invention arepharmaceutically acceptable. They usually include components in additionto the antigens e.g. they typically include one or more pharmaceuticalcarrier(s) and/or excipient(s). As described below, adjuvants may alsobe included. A thorough discussion of such components is available inreference 39.

Vaccine compositions will generally be in aqueous form. However, somevaccines may be in dry form, e.g. in the form of injectable solids ordried or polymerized preparations on a patch.

Vaccine compositions may include preservatives such as thiomersal or2-phenoxyethanol. It is preferred, however, that the vaccine should besubstantially free from (i.e. less than 5 μg/ml) mercurial material e.g.thiomersal-free [30, 40]. Vaccines containing no mercury are morepreferred. α-tocopherol succinate can be included as an alternative tomercurial compounds [30]. Preservative-free vaccines are particularlypreferred.

To control tonicity, it is preferred to include a physiological salt,such as a sodium salt. Sodium chloride (NaCl) is preferred, which may bepresent at between 1 and 20 mg/mi. Other salts that may be presentinclude potassium chloride, potassium dihydrogen phosphate, disodiumphosphate dehydrate, magnesium chloride, calcium chloride, etc.

Vaccine compositions will generally have an osmolality of between 200mOsm/kg and 400 mOsm/kg, preferably between 240-360 mOsm/kg, and willmore preferably fall within the range of 290-310 mOsm/kg. Osmolality haspreviously been reported not to have an impact on pain caused byvaccination [41], but keeping osmolality in this range is neverthelesspreferred.

Vaccine compositions may include one or more buffers. Typical buffersinclude: a phosphate buffer; a Tris buffer; a borate buffer; a succinatebuffer; a histidine buffer (particularly with an aluminum hydroxideadjuvant); or a citrate buffer. Buffers will typically be included inthe 5-20 mM range.

The pH of a vaccine composition will generally be between 5.0 and 8.1,and more typically between 6.0 and 8.0 e.g. 6.5 and 7.5, or between 7.0and 7.8. A process of the invention may therefore include a step ofadjusting the pH of the bulk vaccine prior to packaging.

The vaccine composition is preferably sterile. The vaccine compositionis preferably non-pyrogenic e.g. containing <1 EU (endotoxin unit, astandard measure) per dose, and preferably <0.1 EU per dose. The vaccinecomposition is preferably gluten-free.

Vaccine compositions of the invention may include detergent e.g. apolyoxycthylene sorbitan ester surfactant (known as ‘Tweens’), anoctoxynol (such as octoxynol-9 (Triton X-100) ort-octylphenoxypolyethoxyethanol), a cetyl trimethyl ammonium bromide(‘CTAB’), or sodium deoxycholate, particularly for a split or surfaceantigen vaccine. The detergent may be present only at trace amounts.Thus the vaccine may included less than 1 mg/ml of each of octoxynol-10and polysorbate 80. Other residual components in trace amounts could beantibiotics (e.g. neomycin, kanamycin, polymyxin B).

A vaccine composition may include material for a single immunisation, ormay include material for multiple immunisations (i.e. a ‘multidose’kit). The inclusion of a preservative is preferred in multidosearrangements. As an alternative (or in addition) to including apreservative in multidose compositions, the compositions may becontained in a container having an aseptic adaptor for removal ofmaterial.

Influenza vaccines are typically administered in a dosage volume ofabout 0.5 ml, although a half dose (i.e. about 0.25 ml) may beadministered to children.

Compositions and kits are preferably stored at between 2° C. and 8° C.They should not be frozen. They should ideally be kept out of directlight.

Host Cell DNA

Where virus has been isolated and/or grown on a cell line, it isstandard practice to minimize the amount of residual cell line DNA inthe final vaccine, in order to minimize any oncogenic activity of theDNA.

Thus a vaccine composition prepared according to the inventionpreferably contains less than 10 ng (preferably less than Ing, and morepreferably less than 100 μg) of residual host cell DNA per dose,although trace amounts of host cell DNA may be present.

It is preferred that the average length of any residual host cell DNA isless than 500 bp e.g. less than 400 bp, less than 300 bp, less than 200bp, less than 100 bp, etc.

Contaminating DNA can be removed during vaccine preparation usingstandard purification procedures e.g. chromatography, etc. Removal ofresidual host cell DNA can be enhanced by nuclease treatment e.g. byusing a DNase. A convenient method for reducing host cell DNAcontamination is disclosed in references 42 & 43, involving a two-steptreatment, first using a DNase (e.g. Benzonase), which may be usedduring viral growth, and then a cationic detergent (e.g. CTAB), whichmay be used during virion disruption. Treatment with an alkylatingagent, such as 3-propiolactone, can also be used to remove host cellDNA, and advantageously may also be used to inactivate virions [44].

Adjuvants

Compositions of the invention may advantageously include an adjuvant,which can function to enhance the immune responses (humoral and/orcellular) elicited in a subject who receives the composition. Preferredadjuvants comprise oil-in-water emulsions. Various such adjuvants areknown, and they typically include at least one oil and at least onesurfactant, with the oil(s) and surfactant(s) being biodegradable(metabolisable) and biocompatible. The oil droplets in the emulsion aregenerally less than 5 μm in diameter, and ideally have a sub-microndiameter, with these small sizes being achieved with a microfluidiser toprovide stable emulsions. Droplets with a size less than 220 nm arepreferred as they can be subjected to filter sterilization.

The emulsion can comprise oils such as those from an animal (such asfish) or vegetable source. Sources for vegetable oils include nuts,seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil,the most commonly available, exemplify the nut oils. Jojoba oil can beused e.g. obtained from the jojoba bean. Seed oils include saffloweroil, cottonseed oil, sunflower seed oil, sesame seed oil and the like.In the grain group, corn oil is the most readily available, but the oilof other cereal grains such as wheat, oats, rye, rice, teff, triticaleand the like may also be used. 6-10 carbon fatty acid esters of glyceroland 1,2-propanediol, while not occurring naturally in seed oils, may beprepared by hydrolysis, separation and esterification of the appropriatematerials starting from the nut and seed oils. Fats and oils frommammalian milk are metabolizable and may therefore be used in thepractice of this invention. The procedures for separation, purification,saponification and other means necessary for obtaining pure oils fromanimal sources are well known in the art. Most fish containmetabolizable oils which may be readily recovered. For example, codliver oil, shark liver oils, and whale oil such as spermaceti exemplifyseveral of the fish oils which may be used herein. A number of branchedchain oils are synthesized biochemically in 5-carbon isoprene units andare generally referred to as terpenoids. Shark liver oil contains abranched, unsaturated terpenoids known as squalene,2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene, which isparticularly preferred herein. Squalane, the saturated analog tosqualene, is also a preferred oil. Fish oils, including squalene andsqualane, are readily available from commercial sources or may beobtained by methods known in the art. Another preferred oil isα-tocopherol (see below).

Mixtures of oils can be used.

Surfactants can be classified by their ‘HLB’ (hydrophile/lipophilebalance). Preferred surfactants of the invention have a HLB of at least10, preferably at least 15, and more preferably at least 16. Theinvention can be used with surfactants including, but not limited to:the polyoxyethylene sorbitan esters surfactants (commonly referred to asthe Tweens), especially polysorbate 20 and polysorbate 80; copolymers ofethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO),sold under the DOWFAX™ tradename, such as linear EO/PO block copolymers;octoxynols, which can vary in the number of repeating ethoxy(oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, ort-octylphenoxypolyethoxyethanol) being of particular interest;(octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipidssuch as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such asthe Tergitol™ NP series; polyoxyethylene fatty ethers derived fromlauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants),such as triethyleneglycol monolauryl ether (Brij 30); and sorbitanesters (commonly known as the SPANs), such as sorbitan trioleate (Span85) and sorbitan monolaurate. Non-ionic surfactants are preferred.Preferred surfactants for including in the emulsion are Tween 80(polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate),lecithin and Triton X-100.

Mixtures of surfactants can be used e.g. Tween 80/Span 85 mixtures. Acombination of a polyoxyethylene sorbitan ester such as polyoxyethylenesorbitan monooleate (Tween 80) and an octoxynol such ast-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable. Anotheruseful combination comprises laureth 9 plus a polyoxyethylene sorbitanester and/or an octoxynol.

Preferred amounts of surfactants (% by weight) are: polyoxyethylenesorbitan esters (such as Tween 80) 0.01 to 1%, in particular about 0.1%;octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, or otherdetergents in the Triton series) 0.001 to 0.1%, in particular 0.005 to0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20%, preferably0.1 to 10% and in particular 0.1 to 1% or about 0.5%.

Where the vaccine contains a split virus, it is preferred that itcontains free surfactant in the aqueous phase. This is advantageous asthe free surfactant can exert a ‘splitting effect’ on the antigen,thereby disrupting any unsplit virions and/or virion aggregates thatmight otherwise be present. This can improve the safety of split virusvaccines [45].

Preferred emulsions have an average droplets size of <1 μm e.g. ≦750 nm,≦500 nm, ≦400 nm, ≦300 nm, ≦250 nm, ≦220 nm, ≦200 nm, or smaller. Thesedroplet sizes can conveniently be achieved by techniques such asmicrofluidisation.

Specific oil-in-water emulsion adjuvants useful with the inventioninclude, but are not limited to:

-   -   A submicron emulsion of squalene, Tween 80, and Span 85. The        composition of the emulsion by volume can be about 5% squalene,        about 0.5% polysorbate 80 and about 0.5% Span 85. In weight        terms, these ratios become 4.3% squalene, 0.5% polysorbate 80        and 0.48% Span 85. This adjuvant is known as ‘MF59’ [46-48], as        described in more detail in Chapter 10 of ref. 49 and chapter 12        of ref. 50. The MF59 emulsion advantageously includes citrate        ions e.g. 10 mM sodium citrate buffer.    -   An emulsion of squalene, DL-α-tocopherol, and polysorbate 80        (Tween 80). The emulsion may include phosphate buffered saline.        It may also include Span 85 (e.g. at 1%) and/or lecithin. These        emulsions may have from 2 to 10% squalene, from 2 to 10%        tocopherol and from 0.3 to 3% Tween 80, and the weight ratio of        squalene:tocopherol is preferably ≦1 as this provides a more        stable emulsion. Squalene and Tween 80 may be present volume        ratio of about 5:2 or at a weight ratio of about 11:5. One such        emulsion can be made by dissolving Tween 80 in PBS to give a 2%        solution, then mixing 90 ml of this solution with a mixture of        (5 g of DL-α-tocopherol and 5 m squalene), then microfluidising        the mixture. The resulting emulsion may have submicron oil        droplets e.g. with an average diameter of between 100 and 250        nm, preferably about 180 nm. The emulsion may also include a        3-de-O-acylated monophosphoryl lipid A (3d-MPL). Another useful        emulsion of this type may comprise, per human dose, 0.5-10 mg        squalene, 0.5-11 mg tocopherol, and 0.1-4 mg polysorbate 80        [51].    -   An emulsion of squalene, a tocopherol, and a Triton detergent        (e.g. Triton X-100). The emulsion may also include a 3d-MPL (see        below). The emulsion may contain a phosphate buffer.    -   An emulsion comprising a polysorbate (e.g. polysorbate 80), a        Triton detergent (e.g. Triton X-100) and a tocopherol (e.g. an        α-tocopherol succinate). The emulsion may include these three        components at a mass ratio of about 75:11:10 (e.g. 750 μg/ml        polysorbate 80, 110 μg/ml Triton X-100 and 100 μg/ml        α-tocopherol succinate), and these concentrations should include        any contribution of these components from antigens. The emulsion        may also include squalene. The emulsion may also include a        3d-MPL (see below). The aqueous phase may contain a phosphate        buffer.    -   An emulsion of squalane, polysorbate 80 and poloxamer 401        (“Pluronic™ L121”). The emulsion can be formulated in phosphate        buffered saline, pH 7.4. This emulsion is a useful delivery        vehicle for muramyl dipeptides, and has been used with        threonyl-MDP in the “SAF-1” adjuvant [52] (0.05-1% Thr-MDP, 5%        squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). It can        also be used without the Thr-MDP, as in the “AF” adjuvant [53]        (5% squalane, 1.25% Pluronic L121 and 0.2% polysorbate 80).        Microfluidisation is preferred.    -   An emulsion comprising squalene, an aqueous solvent, a        polyoxyethylene alkyl ether hydrophilic nonionic surfactant        (e.g. polyoxyethylene (12) cetostearyl ether) and a hydrophobic        nonionic surfactant (e.g. a sorbitan ester or mannide ester,        such as sorbitan monoleate or ‘Span 80’). The emulsion is        preferably thermoreversible and/or has at least 90% of the oil        droplets (by volume) with a size less than 200 nm [54]. The        emulsion may also include one or more of: alditol; a        cryoprotective agent (e.g. a sugar, such as dodecylmaltoside        and/or sucrose); aid/or an alkylpolyglycoside. The emulsion may        include a TLR4 agonist [55]. Such emulsions may be lyophilized.    -   An emulsion of squalene, poloxamer 105 and Abil-Care [56]. The        final concentration (weight) of these components in adjuvanted        vaccines are 5% squalene, 4% poloxamer 105 (pluronic polyol) and        2% Abil-Care 85 (Bis-PEG/PPG-16/16 PEG/PPG-16/16 dimethicone;        caprylic/capric triglyceride).    -   An emulsion having from 0.5-50% of an oil, 0.1-10% of a        phospholipid, and 0.05-5% of a non-ionic surfactant. As        described in reference 57, preferred phospholipid components are        phosphatidylcholine, phosphatidylethanolamine,        phosphatidylserine, phosphatidylinositol, phosphatidylglycerol,        phosphatidic acid, sphingomyelin and cardiolipin. Submicron        droplet sizes are advantageous.    -   A submicron oil-in-water emulsion of a non-metabolisable oil        (such as light mineral oil) and at least one surfactant (such as        lecithin, Tween 80 or Span 80). Additives may be included, such        as QuilA saponin, cholesterol, a saponin-lipophile conjugate        (such as GPI-0100, described in reference 58, produced by        addition of aliphatic amine to desacylsaponin via the carboxyl        group of glucuronic acid), dimethyidioctadecylammonium bromide        and/or N,N-dioctadecyl-N,N-bis (2-hydroxyethyl)propanediamine.    -   An emulsion in which a saponin (e.g. QuilA or QS21) and a sterol        (e.g. a cholesterol) are associated as helical micelles [59].    -   An emulsion comprising a mineral oil, a non-ionic lipophilic        ethoxylated fatty alcohol, and a non-ionic hydrophilic        surfactant (e.g. an ethoxylated fatty alcohol and/or        polyoxyethylene-polyoxypropylene block copolymer) [60].    -   An emulsion comprising a mineral oil, a non-ionic hydrophilic        ethoxylated fatty alcohol, and a non-ionic lipophilic surfactant        (e.g. an ethoxylated fatty alcohol and/or        polyoxyethylene-polyoxypropylene block copolymer) [60].

In some embodiments an emulsion may be mixed with antigenextemporaneously, at the time of delivery, and thus the adjuvant andantigen may be kept separately in a packaged or distributed vaccine,ready for final formulation at the time of use. In other embodiments anemulsion is mixed with antigen during manufacture, and thus thecomposition is packaged in a liquid adjuvanted form. The antigen willgenerally be in an aqueous form, such that the vaccine is finallyprepared by mixing two liquids. The volume ratio of the two liquids formixing can vary (e.g. between 5:1 and 1:5) but is generally about 1:1.Where concentrations of components are given in the above descriptionsof specific emulsions, these concentrations are typically for anundiluted composition, and the concentration after mixing with anantigen solution will thus decrease.

Packaging of Vaccine Compositions

Suitable containers for compositions of the invention (or kitcomponents) include vials, syringes (e.g. disposable syringes), nasalsprays, etc. These containers should be sterile.

Where a composition/component is located in a vial, the vial ispreferably made of a glass or plastic material. The vial is preferablysterilized before the composition is added to it. To avoid problems withlatex-sensitive patients, vials are preferably sealed with a latex-freestopper, and the absence of latex in all packaging material ispreferred. The vial may include a single dose of vaccine, or it mayinclude more than one dose (a ‘multidose’ vial) e.g. 10 doses. Preferredvials are made of colourless glass.

A vial can have a cap (e.g. a Luer lock) adapted such that a pre-filledsyringe can be inserted into the cap, the contents of the syringe can beexpelled into the vial (e.g. to reconstitute lyophilised materialtherein), and the contents of the vial can be removed back into thesyringe. After removal of the syringe from the vial, a needle can thenbe attached and the composition can be administered to a patient. Thecap is preferably located inside a seal or cover, such that the seal orcover has to be removed before the cap can be accessed. A vial may havea cap that permits aseptic removal of its contents, particularly formultidose vials.

Where a component is packaged into a syringe, the syringe may have aneedle attached to it. If a needle is not attached, a separate needlemay be supplied with the syringe for assembly and use. Such a needle maybe sheathed. Safety needles are preferred. I-inch 23-gauge, 1-inch25-gauge and ⅝-inch 25-gauge needles are typical. Syringes may beprovided with peel-off labels on which the lot number, influenza seasonand expiration date of the contents may be printed, to facilitate recordkeeping. The plunger in the syringe preferably has a stopper to preventthe plunger from being accidentally removed during aspiration. Thesyringes may have a latex rubber cap and/or plunger. Disposable syringescontain a single dose of vaccine. The syringe will generally have a tipcap to seal the tip prior to attachment of a needle, and the tip cap ispreferably made of a butyl rubber. If the syringe and needle arepackaged separately then the needle is preferably fitted with a butylrubber shield. Preferred syringes are those marketed under the tradename “Tip-Lok”™.

Containers may be marked to show a half-dose volume e.g. to facilitatedelivery to children. For instance, a syringe containing a 0.5 ml dosemay have a mark showing a 0.25 ml volume.

Where a glass container (e.g. a syringe or a vial) is used, then it ispreferred to use a container made from a borosilicate glass rather thanfrom a soda lime glass.

A kit or composition may be packaged (e.g. in the same box) with aleaflet including details of the vaccine e.g. instructions foradministration, details of the antigens within the vaccine, etc. Theinstructions may also contain warnings e.g. to keep a solution ofadrenaline readily available in case of anaphylactic reaction followingvaccination, etc.

Methods of Treatment, and Administration of the Vaccine

The invention provides a vaccine manufactured according to theinvention. These vaccine compositions are suitable for administration tohuman or non-human animal subjects, such as pigs, and the inventionprovides a method of raising an immune response in a subject, comprisingthe step of administering a composition of the invention to the subject.The invention also provides a composition of the invention for use as amedicament, and provides the use of a composition of the invention forthe manufacture of a medicament for raising an immune response in asubject.

The immune response raised by these methods and uses will generallyinclude an antibody response, preferably a protective antibody response.Methods for assessing antibody responses, neutralising capability andprotection after influenza virus vaccination are well known in the art.Human studies have shown that antibody titers against hemagglutinin ofhuman influenza virus are correlated with protection (a serum samplehemagglutination-inhibition titer of about 30-40 gives around 50%protection from infection by a homologous virus) [61]. Antibodyresponses are typically measured by hemagglutination inhibition, bymicroneutralisation, by single radial immunodiffusion (SRID), and/or bysingle radial hemolysis (SRH). These assay techniques are well known inthe art.

Compositions of the invention can be administered in various ways. Themost preferred immunisation route is by intramuscular injection (e.g.into the arm or leg), but other available routes include subcutaneousinjection, intranasal [62-64], oral [65], intradermal [66, 67],transcutaneous, transdermal [68], etc.

Vaccines prepared according to the invention may be used to treat bothchildren and adults. Influenza vaccines are currently recommended foruse in pediatric and adult immunisation, from the age of 6 months. Thusa human subject may be less than 1 year old, 1-5 years old, 5-15 yearsold, 15-55 years old, or at least 55 years old. Preferred subjects forreceiving the vaccines are the elderly (e.g. ≧50 years old, ≧60 yearsold, and preferably ≧65 years), the young (e.g. ≦5 years old),hospitalised subjects, healthcare workers, armed service and militarypersonnel, pregnant women, the chronically ill, immunodeficientsubjects, subjects who have taken an antiviral compound (e.g. anoseltamivir or zanamivir compound; see below) in the 7 days prior toreceiving the vaccine, people with egg allergies and people travellingabroad. The vaccines are not suitable solely for these groups, however,and may be used more generally in a population. For pandemic strains,administration to all age groups is preferred.

Preferred compositions of the invention satisfy 1, 2 or 3 of the CPMPcriteria for efficacy. In adults (18-60 years), these criteria are: (1)≧70% seroprotection; (2) ≧40% seroconversion; and/or (3) a GMT increaseof ≧2.5-fold. In elderly (>60 years), these criteria are: (1) ≧60%seroprotection; (2) ≧30% seroconversion; and/or (3) a GMT increase of≧2-fold. These criteria are based on open label studies with at least 50patients.

Treatment can be by a single dose schedule or a multiple dose schedule.Multiple doses may be used in a primary immunisation schedule and/or ina booster immunisation schedule. In a multiple dose schedule the variousdoses may be given by the same or different routes e.g. a parenteralprime and mucosal boost, a mucosal prime and parenteral boost, etc.Administration of more than one dose (typically two doses) isparticularly useful in immunologically naïve patients e.g. for peoplewho have never received an influenza vaccine before, or for vaccinatingagainst a new HA subtype (as in a pandemic outbreak). Multiple doseswill typically be administered at least 1 week apart (e.g. about 2weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about10 weeks, about 12 weeks, about 16 weeks, etc.).

Vaccines produced by the invention may be administered to patients atsubstantially the same time as (e.g. during the same medicalconsultation or visit to a healthcare professional or vaccinationcentre) other vaccines e.g. at substantially the same time as a measlesvaccine, a mumps vaccine, a rubella vaccine, a MMR vaccine, a varicellavaccine, a MMRV vaccine, a diphtheria vaccine, a tetanus vaccine, apertussis vaccine, a DTP vaccine, a conjugated H. influenzae type bvaccine, an inactivated poliovirus vaccine, a hepatitis B virus vaccine,a meningococcal conjugate vaccine (such as a tetravalent A-C-W135-Yvaccine), a respiratory syncytial virus vaccine, a pneumococcalconjugate vaccine, etc. Administration at substantially the same time asa pneumococcal vaccine and/or a meningococcal vaccine is particularlyuseful in elderly patients.

Similarly, vaccines of the invention may be administered to patients atsubstantially the same time as (e.g. during the same medicalconsultation or visit to a healthcare professional) an antiviralcompound, and in particular an antiviral compound active againstinfluenza virus (e.g. oseltamivir and/or zanamivir). These antiviralsinclude neuraminidase inhibitors, such as a(3R,4R,5S)-4-acetylamino-5-amino-3(1-ethylpropoxy)-1-cyclohexene-1-carboxylicacid or5-(acetylamino)-4-[(aminoiminomethyl)-amino]-2,6-anhydro-3,4,5-trideoxy-D-glycero-D-galactonon-2-enonicacid, including esters thereof (e.g. the ethyl esters) and salts thereof(e.g. the phosphate salts). A preferred antiviral is(3R,4R,5S)-4-acetylamino-5-amino-3(1-ethylpropoxy)-1-cyclohexene-1-carboxylicacid, ethyl ester, phosphate (1:1), also known as oseltamivir phosphate(TAMIFLU™).

General

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x is optional andmeans, for example, x±10%.

Unless specifically stated, a process comprising a step of mixing two ormore components does not require any specific order of mixing. Thuscomponents can be mixed in any order. Where there are three componentsthen two components can be combined with each other, and then thecombination may be combined with the third component, etc.

Where animal (and particularly bovine) materials are used in the cultureof cells, they should be obtained from sources that are free fromtransmissible spongiform encephalopathies (TSEs), and in particular freefrom bovine spongiform encephalopathy (BSE). Overall, it is preferred toculture cells in the total absence of animal-derived materials.

Where a compound is administered to the body as part of a compositionthen that compound may alternatively be replaced by a suitable prodrug.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the expression construct which was used for assayingpol I promoter activity with a luciferase reporter.

FIG. 2 shows the full-length (FL) human pol I promoter sequence (SEQ IDNO: 1). The pHW2000 human Pol I Promoter sequence (SEQ ID NO: 2; “short”human pol I promoter)) within the full-length sequence is shown inunderlined fonts. The arrow indicates the transcriptional start site.

FIG. 3 shows the full-length (FL) canine pol I promoter sequence(NW_(—)878945; SEQ ID NO: 3). The SHORT promoter sequence within thefull-length promoter sequence is shown in underlined capital fonts (SEQID NO: 5); the MID promoter sequence within the full-length promotersequence is shown in underlined capital fonts and bold lowercase fonts(SEQ ID NO: 4);

FIG. 4 shows canine pol I promoter activity in MDCK cells. The solidgrey columns show the results with the FL canine pol I promoter, thecross-hatched columns show the results with the MID canine promoter andthe dotted columns show the results with the SHORT canine pol Ipromoter. “A” indicates LUC and viral polymerase, “B” indicates LUC andinfection (MOI=0.05), “C” indicates LUC and “D” is no DNA. The y-axisindicates relative light units (RLU).

FIG. 5 shows human pol I promoter activity in MDCK 33016 cells. Thesolid grey columns show the results with the human pol I promoter, thecross-hatched columns show the results with the FL canine pol Ipromoter, the dotted columns show the results with the MID canine pol Ipromoter and the vertically hatched columns show the results with theSHORT canine pol I promoter. “A” indicates LUC and viral polymerase, “B”indicates LUC and infection (MOI=0.05) and “C” indicates LUC. The y-axisindicates relative light units (RLU).

FIG. 6 shows a comparison of the activity of the FL and SHORT human polI promoter and the full-length canine pol I promoter in MDCK cells 33016cells. The solid grey columns show the results with the full-lengthhuman promoter, the hatched columns show the results with thefull-length canine promoter and the dotted columns show the results withthe short human pol I promoter. A indicates LUC+polymerase, B indicatesLUC+infection and C shows LUC only. The y-axis indicates relative lightunits (RLU).

FIG. 7A shows the activity of the human pol I promoter (dotted columns)and the canine pol I promoter (cross-hatched columns) in MDCK 33016cells. “X” indicates LUC+polymerase, “Y” indicates LUC+infection and “Z”shows LUC only. The y-axis indicates relative light units (RLU).

FIG. 7B shows the activity of the human pol I promoter (dotted columns)and the canine pol I promoter (cross-hatched columns) in MDCK ATCCcells. “X” indicates LUC+polymerase, “Y” indicates LUC+infection and “Z”shows LUC only. The y-axis indicates relative light units (RLU).

FIG. 8 shows a western blot analysis of M and NP proteins in celllysates after virus rescue.

FIG. 9 shows results of a focus-forming assay using supernatant fromcells infected with reverse genetics constructs.

FIG. 10 shows an alignment of DNA sequences of human and canine pol Ipromoters (SEQ ID NOs 1 and 3, respectively).

FIG. 11A shows the expression levels of a reporter transgene undercontrol of the human pol I (hPolI) promoter or canine pol I promoter(cPolI) in MDCK ATCC, MDCK 33016-PF and 293T cells. The black columnsrepresent the results with 293T cells, the white columns show theresults with MDCK 33016-PF and the cross-hatched columns represent theresults with MDCK ATCC cells.

FIG. 11B compares the transfection efficiency in human and canine cells.The y-axis in both graphs indicates relative light units (RLUs).

FIG. 12A shows the rescue of the A/Puerto Rico/8/34 influenza strain byhuman poll promoter-based reverse genetics in MDCK ATCC, MDCK 33016-PFand 293T cells in the presence (white columns) and absence (blackcolumns) of the TMPRSS2 helper plasmid and with addition of feedercells. The y-axis represents the virus titre (ffu/mL).

FIG. 12B shows the rescue of the A/Puerto Rico/8/34 influenza strain byhuman poll promoter-based reverse genetics in MDCK ATCC, MDCK 33016-PFand 293T cells in the presence (white columns) and absence (blackcolumns) of the TMPRSS2 helper plasmid and without addition of feedercells. The y-axis represents the virus titre (ffu/mL).

FIG. 13 compares the rescue of the A/Puerto Rico/8/34 influenza strainby human or canine pol I-driven reverse genetics in MDCK 33016-PF cells.The black columns show the results in the absence of the TMPRSS2 helperplasmid and the white bars show the result in the presence of theTMPRSS2 helper plasmid. The y-axis represents the virus titre (ffu/mL).

BRIEF DESCRIPTION OF SEQUENCE LISTING

SEQ ID NO: 1 is the full-length (FL) human pol I promoter sequence

SEQ ID NO: 2 is the pHW2000 human Pol I promoter sequence

SEQ ID NO: 3 is the full-length (FL) canine pol I promoter sequence

SEQ ID NO: 4 is the MID canine pol I promoter sequence

SEQ ID NO: 5 is the SHORT canine pol I promoter sequence

SEQ ID NO: 6 is the HA sequence from A/California/04/09

SEQ ID NO: 7 is the HA sequence from A/Chile/1/1983

SEQ ID NO: 8 is the NA sequence from A/California/04/09

SEQ ID NO: 9 is the NA sequence from A/Chile/1/1983

MODES FOR CARRYING OUT THE INVENTION

The human pol I promoter is active in human as well as canine cells.

In order to assess the activity of the pol I promoter in non-endogenoushost cells, MDCK cells were transfected with an expression constructwhich allows expression of a luciferase (luc) RNA in antisense directionunder control of a 487 bp fragment of the human pol I promoter orvarious fragments of the canine pol I promoter (as shown in FIG. 3). Theexpressed RNA can be transcribed into mRNA by a viral polymerase andsubsequently be translated into luc protein. Thus, cells expressing thetransgene can be easily identified by assaying for luciferase activity.In order for the assay to work it is necessary to provide viralpolymerase. This can be achieved by co-transfecting the cell withexpression constructs which encode the viral polymerase or,alternatively, infecting the transfected cell with a helper virus. Theassay is illustrated in FIG. 1.

FIG. 11A shows that the human pol I promoter is able to drive expressionof the transgene in MDCK ATCC cells and also in MDCK 33016-PF cells withthe same efficiency as the canine pol I promoter. The expression levelsof the transgene with the human pol I promoter in MDCK ATCC cells areeven higher than those observed in human 293T cells. In order to confirmthat the transfection efficiency of the tested cell types arecomparable, they were transfected with a construct containing aluciferase gene under control of a CMV promoter. The level of luciferaseactivity was measured. The results are shown in FIG. 11B and confirmthat the transfection efficiency of the tested cells is comparable.

FIG. 4 shows that all tested fragments of the canine pol I promoter candrive expression of the luc transgene in MDCK cells. Furthermore, FIG. 5demonstrates that the full-length human pol I promoter is able to driveexpression of the transgene in MDCK cells and is even more efficientthan the canine pol I promoter.

In order to further define the region of the human pol I promoter whichis necessary to drive expression of the transgene, the experiment wasrepeated with a fragment of the human pol I promoter as shown in FIG. 2(“short” pol I). It was found that, while the full length pol I promoteris more active, the full-length as well as the short human pol Ipromoter are active in MDCK cells (FIG. 6).

The constructs containing the human and canine pol I promoter sequenceswere further transfected into MDCK from ATCC and MDCK 33016 cells [18]in order to determine whether the activity of the human pol I promoteris restricted to a certain cell line. As shown in FIG. 7, the human polI promoter was able to drive expression of the transgene in both celltypes but the expression was more efficient in MDCK 33016 cells.Rescuing influenza virus from MDCK cells using human pol I promoter

The efficiency of influenza virus rescue using the human pol I promoterwas compared in MDCK and 293T cells. The influenza viral genome wascloned into pHW2000 expression vectors [69]. This vector contains afragment of the human pol I promoter which was shown to be active inMDCK cells (see FIG. 5). In particular, the following vectors were used:pHW-WSN PA (0.534 μg/μl); pHW-WSN PB1 (0.432 μg/μl); pHW-WSN PB2 (0.357μg/μl); pHW-WSN NP (0.284 μg/μl); pHW-WSN NS (0.2171 μg/μl); pHW-WSN M(0.232 μg/μl); pHW-WSN HA (0.169 g/μl); pHW-WSN NA (0.280 μg/μl) andpcDNA-TMPRSS (0.775 μg/μl; encoding serine protease). Protein-codinggenes were controlled by a cytomegalovirus (CMV) promoter.

For the virus rescue, 293T cells were seeded at a density of 5×10⁶cells/well in 6-well dishes with 2 ml of Dulbecco's Modified EagleMedium (DMEM) with 10% FCS. MDCK cells were plated at 0.3×10⁶ cell/wellin 6-well dished with 2 ml of medium. The cells were incubated overnightat 37° C. and were transfected when they had reached a confluency of50-80%.

293T and MDCK cells were transfected using FuGENE 6 Transfection Reagent(Roche Cat.#11988387001) and Lipofectamine LTX Plus Reagent (InvitrogenCat.#15338-100), respectively. The cells were transfected with 1 μg ofeach vector in accordance with the following protocols. For FuGENE 6,the reagent (3 μl of FuGENE/μg DNA) was diluted in 73 μl serum-freemedium (without antibiotics), mixed gently and incubated at roomtemperature for 5 minutes. Afterwards the DNA was added to each to thediluted FuGENE, mixed gently and incubated at room temperature for atleast 15 minutes. The DNA/FuGENE complex was added drop wise to the 293Tcells without removing the growth medium and the cells were incubated at37° C. for 24 hours.

For transfection with lipofectamine, the reagent (25 μl) was diluted in500 μl serum-free medium and incubated at room temperature for 5minutes. The DNA was added and the mixture was incubated at roomtemperature for 30 minutes. Following incubation, 500 μl of serum-freemedium was added drop wise to the transfection reagent after the growthmedium had been removed from the cells. The cells were subsequentlyincubated at 37° C. for 24 hours. 24 hours after transfection, themedium was changed.

Two days after infection, the supernatant from the cells was collectedby centrifugation at 1000 rpm for 5 minutes. The virus collected in thesupernatant was used for Focus-Forming Assays. Furthermore, the infectedcells were lysed and used for Western Blot analysis.

Western Blot Analysis

The 293T and MDCK cells were lysed and subjected to Western Blotanalysis in accordance with standard protocols. Antibodies against the Mand NP protein were used to detect these proteins on the membrane.Antibodies against S6 were used as a loading control. The lanes labelledas ‘WSN’ were loaded with proteins from the rescued virus. The laneslabelled ‘M’ and ‘NP’ contain recombinant M and NP proteins as acontrol. As these recombinant proteins were expressed from a differentgene they migrate slightly slower in the gel.

The results of the analysis are shown in FIG. 8 where it is evident thatthe expression construct under control of the human pol I promoterallows viral rescue in 293T as well as in MDCK cells.

Focus-Forming Assays

Uninfected MDCK cells were plated at a density of 6.25×10⁴ cells/well in48 well plates in 500 μl of DMEM with 10% FCS. The next day cells wereinfected with viruses in a volume of 100-150 μl for 2 hours at 37° C.The cells were thereby infected with various dilutions of the virus. Twohours post-infection, the medium was aspirated and 500 μl of DMEM with10% PCS was added to each well. The cells were incubated at 37° C. untilthe next day.

24 hours after infection, the medium was aspirated and the cells washedonce with PBS, 500 μl of ice-cold 80% acetone in PBS was added to eachwell followed by incubation at 4° C. for 30 minutes. The acetone mix wasaspirated and the cells washed once with PBST (PBS+0.1% Tween). 500 μlof 2% BSA in PBS was added to each well followed by incubation at roomtemperature (RI) for 30 minutes. 500 μl of a 1:6000 dilution of anti-NPwas added in blocking buffer followed by incubation at RT for 2 hours.The antibody solution was aspirated and the cells washed twice withPBST. Secondary antibody (goat anti mouse) was added at a dilution1:2000 in 500 μl blocking buffer and the plate was incubated at RT for 2hours. The antibody solution was aspirated and the cells washed threetimes with PBST. 500 μl of KPL True Blue was added to each well andincubated for 10 minutes. The reaction was stopped by aspirating theTrue-Blue and washing once with dH₂O. The water was aspirated and thecells left to dry.

The results of the assay are shown in FIG. 9 which demonstrates clearlythat infectious virus was obtained from 293T as well as MDCK cells.

Virus rescue of the A/Puerto Rico/8/34 influenza strain using a humanpol I reverse genetics system was also tested in MDCK ATCC, MDCK33016-PF and 293T cells as described in reference 70. Experiments wereperformed in which the virus was rescued in the presence and absence ofthe helper plasmid TMPRSS2 which encodes a serine protease. Furthermore,the viral rescue was performed with and without the addition of feedercells 24 hours after the viral rescue. The results are shown in FIG. 12and demonstrate that efficient viral rescue could be achieved in MDCKcells under various conditions using the human pol I promoter.

To compare whether the efficiency of viral rescue in MDCK 33016-PF usinga human poll promoter is comparable with the rescue using a canine pol Ipromoter, the cells were transfected with a human pol I RG system or acanine pol I RG system as described in reference 70. The experimentswere performed in the presence and absence of the TMPRSS helper plasmid.The results (FIG. 13) demonstrate that the A/Puerto Rico/8/34 strain wasrescued with comparable efficiency to the canine pol I system when thehuman pol I system was used.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

REFERENCES

-   [1] WO2007/002008-   [2] WO2007/124327-   [3] Koudstaal et al. (2009) Vaccine 272588-2593-   [4] WO2009/000891-   [5] Sambrook et al, Molecular Cloning: A Laboratory Manual, 2 ed.,    1989, Cold Spring Harbor Press, Cold Spring Harbor, N. Y-   [6] Racaniello and Baltimore (1981) Science 214:916-919-   [7] Kaplan et al. (1985) Proc Natl Acad Sci USA 82: 8424-8428-   [8] Fodor et al. (1999) J. Virol 73(11):9679-9682-   [9] Hoffmann et al. (2002) Proc Natl Acad Sci USA 99: 11411-11416-   [10] Kobayashi et al. (2007) Cell Host Microbe 19; 1(2):147-57-   [11] Stech et al. (2008) Nucleic Acids Res 36(21):e139-   [12] Kistner et al. (1998) Vaccine 16:960-8-   [13] Kistner et al. (1999) Dev Biol Stand 98:101-110-   [14] Bruhl et al. (2000) Vaccine 19:1149-58-   [15] Pau et al. (2001) Vaccine 19:2716-21-   [16] http//www.atcc.org/-   [17] http/locus.umdnj.edu/-   [18] WO97/37000-   [19] Brands et al. (1999) Dev Biol Stand 98:93-100-   [20] Halperin et al. (2002) Vaccine 20:1240-7-   [21] EP-A-1260581 (WO01/64846)-   [22] WO2006/071563-   [23] WO2005/113758-   [24] WO97/37001-   [25] Neumann et al. (2005) Proc Natl Acad Sci USA 102: 16825-9-   [26] WO02/28422-   [27] WO02/067983-   [28] WO02/074336-   [29] WO01/21151-   [30] WO02/097072-   [31] WO2005/113756-   [32] Vaccines. (eds. Plotkins & Orenstein). 4th edition, 2004, ISBN:    0-7216-9688-0-   [33] Huckriede et. al. (2003) Methods Enzymol 373:74-91-   [34] Treanor et al. (1996) J Infect Dis 173:1467-70-   [35] Keitel et al. (1996) Clin Diagn Lab Immunol 3:507-10-   [36] Herlocher et al. (2004) J Infect Dis 190(9): 1627-30-   [37] Le et al (2005) Nature 437(7062):1108-   [38] WO20081068631-   [39] Gennaro (2000) Remington: The Science and Practice of Pharmacy.    20th edition, ISBN: 0683306472-   [40] Banzhoff (2000) Immunology Letters 71:91-96-   [41] Nony et al. (2001) Vaccine 27:3645-51-   [42] EP-B-0870508-   [43] U.S. Pat. No. 5,948,410-   [44] WO2007/052163-   [45] WO2007/052061-   [46] WO90/14837-   [47] Podda & Del Giudice (2003) Expert Rev Vaccines 2:197-203-   [48] Podda (2001) Vaccine 19: 2673-2680-   [49] Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell    & Newman) Plenum Press 1995 (ISBN 0-306-44867-X)-   [50] Vaccine Adjuvants: Preparation Methods and Research Protocols    (Volume 42 of Methods in Molecular Medicine series). ISBN:    1-59259-083-7. Ed. O'Hagan-   [51] WO2008/043774-   [52] Allison & Byars (1992) Res Immunol 143:519-25-   [53] Hariharan et al. (1995) Cancer Res 55:3486-9-   [54] US-2007/014805-   [55] US-2007/0191314-   [56] Suli et al. (2004) Vaccine 22(25-26):3464-9-   [57] WO95/11700-   [58] U.S. Pat. No. 6,080,725-   [59] WO02005/097181-   [60] WO2006/13373-   [61] Potter & Oxford (1979) Br Med Bull 35:69-75-   [62] Greenbaum et at (2004) Vaccine 22:2566-77-   [63] Zurbriggen et al. (2003) Expert Rev Vaccines 2:295-304-   [64] Piascik (2003) J Am Pharm Assoc (Wash DC). 43:728-30-   [65] Mann et at (2004) Vaccine 22:2425-9-   [66] Halperin et al. (1979) Am J Public Health 69:1247-50-   [67] Herbert et al. (1979) J Infect Dis 140:234-8-   [68] Chen et al. (2003) Vaccine 21:2830-6-   [69] Hoffmann et al. (2000) Proc Natl Acad Sci USA 97:6108-   [70] Suphaphiphat et al. (2010) J. Virol. 84(7) 3721-3725

1: A host cell comprising at least one expression construct encoding aviral RNA molecule, wherein expression of the viral RNA molecule fromthe construct is controlled by a pol I promoter which is not endogenousto the host cell's taxonomic order. 2: A cell having at least oneendogenous pol I promoter which control(s) expression of endogenous rRNAand at least one non-endogenous pol I promoter which control(s)expression of a viral RNA or the complement thereof. 3: A method forproducing a recombinant virus, comprising a step of growing the cell ofclaim 1 under conditions where the viral RNA molecule is expressed inorder to produce virus. 4: The method of claim 3 further comprisinginfecting a culture host with the virus produced by growing the cell;culturing the culture host infected with the virus to produce furthervirus; and purifying the further virus obtained by culturing theinfected culture host. 5: The method of claim 4 further comprisingpreparing a vaccine from the purified further virus. 6: The cell ofclaim 1, wherein the pol I promoter is a primate pol I promoter and thecell is a non-primate cell. 7: The cell of claim 1, wherein the pol Ipromoter is a non-canine pol I promoter and the cell is a canine cell.8: The cell of claim 7 wherein the pol I promoter is a human pol Ipromoter. 9: The cell of claim 8 wherein the cell is an MDCK cell. 10:The cell of claim 9 wherein the MDCK cell is cell line MDCK 33016 (DSMACC2219). 11: The cell of claim 1, wherein the cell includes at leastone bi-directional expression construct. 12: The cell of claim 1,wherein the expression construct is an expression vector or a linearexpression construct. 13: The cell of claim 1, wherein the virus is asegmented virus. 14: The cell of claim 1, wherein the virus is anon-segmented virus. 15: The cell of claim 1, wherein the virus is anegative-strand RNA virus. 16: The cell of claim 15, wherein the virusis influenza virus.